Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, based on the embodiments of the invention, which are apparent to those of ordinary skill in the art without inventive faculty, are intended to be within the scope of the invention. Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, based on the embodiments of the invention, which are apparent to those of ordinary skill in the art without inventive faculty, are intended to be within the scope of the invention.
The embodiment of the invention provides a preparation method of a conductive hydrogel dressing, which specifically comprises the steps of fully mixing and reacting a plurality of components such as a high polymer material with good biocompatibility, borax, tannic acid, keratin and the like, and finally preparing the hydrogel dressing with excellent conductive performance. In the preparation process, borate ions and sodium ions in borax play a key role. Wherein, one part of borate ions and hydroxyl groups of biocompatible polymer materials, such as polyvinyl alcohol, form stable borate bonds through chemical reaction, thereby realizing the cross-linking structure of the material, and the other part of borate ions keep a free state. At the same time, a large amount of sodium ions are also present in the colloid in a free manner, and the free ions together give the hydrogel dressing good electrical conductivity.
The conductive hydrogel dressing not only has excellent conductive characteristics, but also can be effectively combined with an electric stimulation therapy, and the healing process of wounds is further promoted through the effect of electric stimulation. In addition, the dressing can also sense and transmit electrophysiological signals of a wound area, so that the real-time monitoring of the wound healing process is realized.
In some embodiments, the biocompatible polymeric material is selected from a variety of materials including, but not limited to, polyvinyl alcohol, polyacrylic acid, various derivatives of polyvinyl alcohol, and various derivatives of polyacrylic acid. By this selection, the safety and applicability of the material in the biological environment are ensured.
In other specific embodiments, the biocompatible polymeric material is polyvinyl alcohol. The material becomes an ideal choice object due to the excellent biocompatibility, good mechanical property and easy processing property, and ensures the high efficiency and reliability in practical application.
In some embodiments, the keratin selected is derived primarily from wool. The wool is used as a natural polymer material, the keratin component of the wool has unique biocompatibility and biodegradability, simultaneously, mercapto (-SH) and negative charge groups which are rich in the surface of the wool can specifically adsorb blood coagulation factor XII and platelets, and the blood coagulation cascade reaction is accelerated through the electrostatic effect, so that the hemostatic efficiency and the biocompatibility of the material are improved, and the keratin source is wide, is cheap and easy to obtain, and has a relatively wide application prospect in the field of biomedical materials.
In some specific embodiments, the biocompatible polymeric material is set to a mass percentage ranging from 5% to 10% in the overall formulation, which is intended to ensure the safety and stability of the material in a biological environment. Meanwhile, the mass percentage of the tannic acid in the formula is controlled to be between 0.25 and 2 percent, and the range is set for fully playing the antioxidant and antibacterial roles of the tannic acid. In addition, the mass percentage of the keratin in the formula is also limited to the range of 0.25-2%, and the addition of the keratin helps to improve the mechanical strength and biocompatibility of the material. Furthermore, the mass percentage of the borax in the formula is set to be 0.5-2%, and the existence of the borax can effectively adjust the ion conductivity of the material. The materials obtained by combining the proportions of the components exhibit excellent conductivity.
In other embodiments, the polyvinyl alcohol is precisely 6.25% by weight of the overall formulation, and this ratio of polyvinyl alcohol provides good substrate support and adhesion. Meanwhile, the weight percentage of the tannic acid in the formula is determined to be 0.75%, and the tannic acid with the concentration can effectively exert the antioxidation and antibacterial properties. In addition, the mass percentage of the keratin in the formula is set to be 1.25%, and the keratin in the proportion contributes to further improving the mechanical property and biocompatibility of the material. Furthermore, the mass percentage of the borax in the formula is precisely controlled to be 0.75%, and the borax with the concentration can effectively adjust the ion conductivity of the material. By these precise proportioning designs, the resulting materials also exhibit excellent electrical conductivity.
In particular embodiments, to ensure optimal mixing and adequate chemical reaction of the components, it is necessary to place the components in a temperature-controlled environment within a temperature range of precisely 20 to 30 degrees celsius during operation. The temperature interval is not only beneficial to the molecular movement among the components to reach a proper activity degree and promote the effective contact and interaction among the components, but also can effectively avoid adverse effects such as slow reaction caused by too low temperature or component decomposition caused by too high temperature, thereby ensuring the smooth progress of the whole mixing and reaction process and the quality and stability of the final product.
The embodiment of the invention aims to provide a conductive hydrogel dressing prepared by a specific method. The conductive hydrogel dressing adopts unique technology and technique in the preparation process, ensures good conductive performance and biocompatibility, and is suitable for application in various scenes.
The embodiment of the invention also provides a device integrating the healing promotion and process monitoring functions. The core component of the device is the conductive hydrogel dressing. By combining the conductive hydrogel dressing with an advanced electronic monitoring technology, the device not only can effectively promote the healing process of wounds, but also can monitor the healing progress in real time.
The embodiment of the invention provides a non-disease treatment method for electrically promoting healing, which is characterized in that a conductive hydrogel dressing is connected with an electric stimulation device, and specific mode electric stimulation with a certain time is applied by controlling the intensity of current at intervals of a certain time. The method can promote cell migration and promote wound healing. The dressing is designed to be matched with the electrical characteristics of human skin so as to achieve the optimal electrical stimulation treatment effect.
In some specific embodiments, the specific pattern of electrical stimulation encompasses multiple types including, but not limited to, a constant current direct current stimulation, a constant voltage direct current stimulation, an alternating current stimulation, and a pulsed electrical stimulation. These different types of electrical stimulation each have unique application scenarios and effects.
In some embodiments, the specific parameter range of the constant current direct current stimulation is set to 0.3 to 1.0 milliamp (mA), and the current intensity within this range can ensure safety and effectively promote migration and proliferation of cells. Likewise, the parameter range of the constant voltage direct current stimulation is set to 1.5 to 6.0 volts (V), which is also in a safe and controllable range, and has been confirmed to have a certain effect of promoting cell migration and proliferation.
Furthermore, in other embodiments, the parameters of the alternating current stimulation are set to a current intensity between 0.3 and 1.0 milliamp (mA) and a frequency range between 5 and 20 hertz (Hz). This mode of alternating current stimulation can play a positive role under different physiological and pathological conditions. The parameters of the pulsed electrical stimulation are set to a frequency range between 25 and 100 hertz (Hz), which high frequency pulsed electrical stimulation exhibits significant biological effects in certain applications. By precisely controlling the electrical stimulation parameters, the regulation and control effects on the cell behaviors can be maximally exerted on the premise of ensuring safety.
In some specific embodiments, a target cell for which a non-disease therapeutic method that utilizes electrical stimulation to promote healing encompasses a variety of cell types including, but not limited to, mouse fibroblasts, mouse myoblasts, and human umbilical vein endothelial cells. These cells may be used alone or in combination of a plurality of cells to achieve the optimal healing promoting effect.
Likewise, in some detailed embodiments, a target wound animal model for which a non-disease treatment method that promotes healing by means of electrical stimulation is applicable also includes a plurality of different wound types. These models include, but are not limited to, skin wounds of rats and mice, diabetic ulcers of rats and mice, wounds at the locomotor joints of rats and mice, skin wounds of rabbit ears, infected wounds of rats and mice, subcutaneous abscesses of rats and mice, and the like. These wound models may be of a single type or may be used in combination of multiple types to more fully assess and verify the effectiveness and safety of the electrical healing-promoting method.
In some embodiments, when the cell type of interest selected for the experiment is mouse fibroblast (L929) or Human Umbilical Vein Endothelial Cells (HUVEC), the parameters for constant DC stimulation used were set to 0.5 mA for optimal cell migration. This parameter was verified by several experiments and was considered to be the most suitable electrical stimulation intensity for both cell types. When the target cell type is changed into mouse myoblasts (C2C 12), the parameters of the constant direct current stimulation are adjusted to be 0.8 mA correspondingly in order to achieve the best cell migration effect. The setting of this parameter is also based on a large amount of experimental data, ensuring that the mouse myoblasts are able to exhibit optimal migration capacity at the electrical stimulation intensity.
In other embodiments, the interval is strictly controlled between 24 and 48 hours, and the duration of the electrical stimulation is set to 10 to 20 minutes. Specifically, under certain experimental conditions, the time of electrical stimulation was precisely set to 15 minutes. The time range is selected by taking the influence of the electrical stimulation on the cell migration effect into consideration, and the practical feasibility and safety of experimental operation are also considered. Through multiple experiments, the parameter range can not only remarkably improve the migration efficiency of cells, but also has higher safety, and ensures that unnecessary damage to the cells is not caused in the experimental process.
Embodiments of the present invention provide a non-disease diagnostic method for monitoring wound healing processes. The method is characterized in that a special conductive hydrogel dressing is applied to a wound area, and the real-time tracking of the wound healing process is realized by accurately monitoring the change of an electric signal generated by joints near the wound area in the motion process. During specific operation, cover dressing in wound area, this dressing possesses self-adaptation characteristic, can carry out the self-adjustment according to the specific shape of wound, ensures that dressing and surface of a wound reach the state of laminating completely to improve the accuracy and the validity of monitoring.
During monitoring, the range of variation of the electrical resistance signal of the wound is set to be greater than 10%, which is significantly different from the range of variation of the electrical resistance signal of normal skin or healed wound, which is typically between 5% and 10%. Whereas for skin whose surface appears to heal but there is still damage inside, the range of variation in the resistance signal is less than 5%. Based on the difference of the resistance signal changes, the scheme provided by the invention can be adopted to accurately monitor the wound healing process and judge whether the wound heals well.
In some specific embodiments, the amplitude of motion of the joint near the wound area is controlled between 30 and 60 degrees for further optimizing the monitoring effect. In other embodiments, the amplitude of the motion of the joint is precisely set to 45 degrees. Through these careful parameter settings, changes in the electrical signal can be more effectively captured, thereby providing more reliable data support for monitoring wound healing.
Example 1
A conductive hydrogel wound dressing integrating healing promotion and progress monitoring functions is shown in figure 1.
(1) Firstly, 40 g wool is cut, washed 2 times and soaked in a mixed solution of 1: 1L ethanol and acetone (volume ratio is 1:1). Stirring at 200 rpm/min for 2 hours by using a homogenizer, washing with deionized water for 4 times, and drying in a 60 ℃ oven overnight to obtain defatted wool. Next, 5 g defatted wool was dissolved in 50 aqueous solution mL containing 6g sodium sulfide, 24 g urea, and 1.442 g Sodium Dodecyl Sulfate (SDS), heated to 60 ℃ in a water bath and stirred for 8 hours to prepare a crude keratin extract. After trimming the crude keratin extract, placing in a centrifuge at 11000 rpm/min, and centrifuging for 30 min to remove completely dissolved wool. Pouring the supernatant after centrifugation into a 3500D dialysis bag for dialysis purification, intercepting a dialysis membrane with proper length, filling into a crude keratin solution, putting into deionized water with the volume ratio of about 1:100 for dialysis for 3 days, and replacing water every 6 hours. After the dialysis is completed, the keratin solution is frozen overnight and then put into a freeze dryer for freeze drying, thus obtaining keratin powder.
(2) Next, 0.5 g polyvinyl alcohol (PVA, P) was weighed, 3: 3 mL ultrapure water was added thereto, and stirred at 95℃for 1 hour to completely dissolve the polyvinyl alcohol, thereby obtaining a PVA solution. 0.1 g keratin (W) was weighed and dissolved in 2 mL ultrapure water, and after sufficient dissolution, it was added to the PVA solution and stirred slowly at room temperature for 1 hour. Then 0.06 g borax (B) and 0.06 g tannic acid (T) are dissolved in 3 mL ultrapure water. After the mixed solution of PVA and keratin is stirred slowly for 5 seconds, the ion-conducting hydrogel dressing is formed.
Comparative example 1
Mixing and stirring polyvinyl alcohol (P) and borax (B) to prepare the ion-conductive hydrogel dressing (PB), and carrying out the rest operation steps in the same manner as in example 1.
Comparative example 2
Polyvinyl alcohol (P), keratin (W) and borax (B) are mixed and stirred to prepare the ion-conductive hydrogel dressing (PWB), and the rest operation steps are the same as in example 1.
Comparative example 3
The polyvinyl alcohol (P), tannic acid (T) and borax (B) are mixed and stirred to prepare the ion conductive hydrogel dressing (PTB), and the rest operation steps are the same as in example 1.
Comparative example 4
In this example 1, the mass percentages of the other components were fixed, and the proportions of the individual components were adjusted, respectively, to form an ion-conductive hydrogel dressing, and the remaining operation steps were the same as in example 1.
Comparative example 5
The polyvinyl alcohol (P), the silk fibroin (S), the tannic acid (T) and the borax (B) are mixed and stirred to prepare the ion-conductive hydrogel dressing (PSB), and the rest operation steps are the same as in example 1.
The electrically conductive hydrogels prepared in this example 1 and comparative examples 1 to 5 were characterized for electrical properties and biocompatibility, and the results are shown in fig. 5 to 7.
The electrical property test is to standardize the appearance of the sample by a 3D printing mould (size: 30 mm multiplied by 10mm multiplied by 10 mm), and high-purity copper foil electrodes are attached to the two ends of the mould, so that the contact area of the electrode-gel interface is ensured to be constant. The conductive hydrogels prepared in this example 1 and comparative examples 1 to 5 were placed in a cavity of a mold and completely adhered to the mold, and a precision source measuring unit (TH 1991B, economical electronics, jiangsu, china) was connected to measure the direct current resistance value (R), the sampling interval was 60 seconds, and the average value of three measurements was taken, and the conductivity (σ) was calculated from formula (1) based on ohm's law:
wherein L is the electrode spacing and A is the effective conductive cross-sectional area.
The biocompatibility test is that the conductive hydrogels prepared in this example 1 and comparative examples 1-3 were leached 24 h using complete culture at 37 ℃ for later use through cytotoxicity test. L929 cells in the logarithmic growth phase were inoculated into 96-well plates at a density of 1X 104 cells/well, and after the cells were attached (about 6 h), 100. Mu.L of the control medium (n=5 per group) of the hydrogel extracts of example 1 and comparative examples 1 to 3 (PB, PWB, PTB, PWTB, concentration 1 mg/mL) was added, respectively, after the original medium was discarded. After further culturing the cells 24, 48 and 72 h, 10. Mu.L of CCK-8 reagent was added to each well and incubated 2h in the dark. Absorbance (OD value) was measured at wavelength 450 nm using a microplate reader, where the blank is a well containing only CCK-8 reagent and medium. The cell viability was calculated according to equation (2) and the biocompatibility of the materials was compared.
The hydrogel in fig. 5 (a) shows that the composite hydrogel doped with wool keratin and tannic acid has higher conductivity than the conductive hydrogel of a single component and similar to the conductivity of human skin, as can be seen from fig. 5 (B), the conductivity also changes with the strain, and when the hydrogel is cut, the circuit is interrupted and the LED indicator is turned off. The two sections are then contacted 24h to effect a self-healing process, and the LED indicator lights on the circuit are lit again.
The mass percentage of the other components in the embodiment 1 is fixed, the proportion of the single components is respectively adjusted, and the conductivity of the corresponding conductive hydrogel is tested. As can be seen from fig. 6, the conductivity of the hydrogel was different by adjusting the proportions of polyvinyl alcohol, borax, tannic acid and keratin, and it can be seen from the results that the conductivity of the hydrogel was optimal when the mass percentage of polyvinyl alcohol was 6.25%, the mass percentage of tannic acid was 0.75%, the mass percentage of keratin was 1.25% and the mass percentage of borax was 0.75%. This is because tannic acid can combine with components in the hydrogel through hydrogen bonds or other chemical bonds, changing the network structure, making ions more likely to migrate in the network, and improving conductivity. And tannic acid and keratin are subjected to hydrogen bond and ionic bond, so that the stability and solubility of the keratin are improved, and meanwhile, the conductivity of the hydrogel is also improved. However, too high a concentration of tannins can denature the keratin to form flocculent precipitates, thereby impeding migration of ions in the hydrogel system.
Taking fresh anticoagulated whole mouse blood, centrifuging for 15 min at 3000 rpm, discarding the upper plasma and pale yellow layer, retaining the bottom erythrocyte pellet, re-suspending the erythrocyte pellet with equal volume of phosphate buffer saline (PBS, pH 7.4), and repeating the centrifugation until the supernatant is colorless and transparent to obtain purified erythrocyte suspension. The hydrogels prepared in this example 1 and comparative examples 1-3 were each placed in a 1.5 mL centrifuge tube, each tube was added with 1mL of 2% RBC suspension (PBS dilution), incubated at 37℃for 4 hours, with the blank set being the PBS-diluted 2% RBC suspension and the positive set being the deionized water-diluted 2% RBC suspension. After the end of incubation, each tube was centrifuged at 3000 rpm for 10 minutes, 100 μl of supernatant was transferred to a 96-well plate, and absorbance was measured at 540: 540 nm wavelength using a microplate reader, and 4 wells (n=4) were set per group. The haemolysis rate of each group was calculated according to formula (3):
As can be seen from the results of (A) in FIG. 7, the hemolysis rate of each component hydrogel was less than 5%, which meets the relevant safety standards. From the results of the cell compatibility in (B-C) of fig. 7, it can be seen that the cell proliferation gradually increased in 24 hours, 48 hours and 72 hours, and that the proliferation level at 72 hours was significantly higher in PWTB groups compared to the control group. The live/dead staining results also showed that green fluorescence intensity increased with time, indicating that cell viability increased gradually with increasing culture time. PWTB the hydrogel group showed a significantly stronger green fluorescence signal than the control group, indicating that the hydrogel had a positive promoting effect on cell survival and proliferation.
The hydrogel of this example 1 was evaluated for in vivo hemostatic performance by a rat liver hemorrhage model, and after anesthetizing the animal, the abdomen was cut to expose the liver. A syringe needle was used to perform a liver puncture on the liver, and then a weighed filter paper was placed under the liver. The hydrogel is then rapidly infused or adhered to the bleeding site and the weight of the blood loss is recorded over a set period of time. The untreated wound served as a control.
From the results shown in FIG. 7 (D-F), it can be seen from the data that the group to which the hydrogel of the present example was applied was able to rapidly stop bleeding in a short time, as compared with the group not treated, and the bleeding amount was much lower than that of the blank group. The hydrogels of this example 1, comparative examples 1-3 and comparative example 5 were subjected to a coagulation index (BCI) test, and 0.1M CaCl2 was added to heparinized mouse blood at a ratio of 10:1 to activate the blood. mu.L of activated blood was placed on the hydrogel surface and the non-coagulated blood was dissolved at various time points using 5 mL deionized water. The OD value at 540 nm was tested by a microplate reader. 50. mu.L of blood in 5 mL deionized water served as a control. BCI is calculated by the following equation 4:
The lower the BCI, the more effectively the blood coagulation can be stimulated. As can be seen from the results of (G) in fig. 7, the above data of hemostasis, which is superior to that of comparative example 1 of the basic gel-forming component, is probably due to the fact that keratin has abundant thiol groups (-SH) and disulfide bonds (-S-), can promote cell adhesion and tissue repair, and helps coagulation and wound healing, and simultaneously, the keratin material can promote platelet adhesion and aggregation by interacting with platelets under physiological environment, thereby accelerating the hemostasis process. The single-component tannic acid accelerates the formation of blood clots mainly through protein precipitation and vasoconstriction, but when the concentration of tannic acid is too high, the tannic acid possibly has cytotoxicity to lead to the inactivation of blood platelets, and the hemostatic effect is affected. Compared with comparative examples 2 and 3 with single components, keratin can form hydrogen bond and covalent crosslinking with tannic acid, so that PWTB hydrogel forms a denser and stronger network structure, and meanwhile, the embodiment cooperates with various mechanisms such as protein adhesion, blood coagulation, vasoconstriction, hydrogel barrier and the like of keratin and tannic acid, and can effectively promote hemostasis. It can be observed by comparing silk fibroin, which is a natural polymer material similar to keratin, can promote tissue regeneration and cell adhesion, and the structure of the silk fibroin is favorable for forming a stable barrier to assist hemostasis, but the procoagulant effect is not obvious, and the result is similar to that of the basic gel-forming component comparative example 1.
Example 2
The scheme of electrically stimulating cells using the conductive hydrogel prepared in example 1 is shown in fig. 2, and the specific procedure is as follows:
(1) Parallel lines were drawn on the bottom of the 12-well plate using a mark pen, ensuring that the parallel line spacing was 0.5-1 cm. L929 cells in the logarithmic growth phase are digested into single cell suspension by trypsin, inoculated into 12-well plates, and 4 x 10 times 4 cells are respectively planted in each well. After addition of complete medium, the cells were incubated in a 37℃5% CO2 incubator for 24 hours until the cells completely covered the bottom of the well plate.
(2) Scratches were made along a ruler with a 10 μl gun head, the direction of the scratches being perpendicular to the marking line. A platinum wire of 0.2 mm was selected as a wire, placed in a culture medium, and fixed to the edge of the well plate with an insulating tape.
(3) The SS-L303SPD linear power supply is used for connecting two ends of the lead, and electric stimulation is performed. The electrical stimulation was performed by applying a constant current of 1.5V, 3.0V, 4.5V, 6.0V, respectively, with 2 replicate wells per group, 15 minutes per day of electrical stimulation, during which time the culture was performed with DMEM medium. Cell migration was observed at 0h, 24h, 48 h, 72 h, respectively.
The cells electrically stimulated in this example were observed under a microscope and quantitatively analyzed. It can be seen from the cell scratch experiments that the electrical stimulation has a remarkable effect on promoting migration of L929 cells. According to FIG. 8A, it is shown that at 72 h th, the cell migration effect under 4.5V electrical stimulation is optimal and the scratch area is minimal.
Example 3
The scheme of electrically stimulating cells using the conductive hydrogel prepared in example 1 is shown in fig. 2, and the specific procedure is as follows:
(1) Parallel lines were drawn on the bottom of the 12-well plate using a mark pen, and the parallel line pitch was controlled between 0.5 and 1 cm. HUVEC cells in the logarithmic growth phase were trypsinized into single cell suspensions and inoculated into 12-well plates, each of which was seeded with 4X 10≡4 cells. After addition of complete medium, the cells were incubated in a 37℃5% CO2 incubator for 24 hours until the bottom of the well plate was completely confluent.
(2) Scratches were made along a ruler with a10 μl gun head, the direction of the scratches being perpendicular to the marking lines. A platinum wire of 0.2mm was used as a wire and placed in a culture medium and fixed to the edge of the well plate with an insulating tape.
(3) And (3) connecting the two ends of the lead by adopting an SS-L303SPD linear power supply to perform electric stimulation. Both ends of the wire were electrically stimulated with a constant current of 1.5V, 3.0V, 4.5V, 6.0V, respectively, 2 duplicate wells were placed in each group, each group was electrically stimulated for 15 minutes per day, during which time culture was performed with DMEM medium, and cell migration was observed at 0h, 24h, 48 h, respectively.
The cells electrically stimulated in this example were observed under a microscope and quantitatively analyzed. As can be seen from the cell scratch experiments, the electrical stimulation has a certain effect on promoting the migration of HUVEC cells. According to FIG. 8B, it is shown that at 48 h, group 4.5V showed the best cell migration effect and the scratch was substantially healed.
Example 4
The scheme of electrically stimulating cells using the conductive hydrogel prepared in example 1 is shown in fig. 2, and the specific procedure is as follows:
(1) Parallel lines were drawn on the bottom of the 12-well plate using a mark pen, with the line spacing controlled between 0.5-1 cm. C2C12 cells in the logarithmic growth phase were trypsinized into single cell suspensions and seeded in 12 well plates with 4 x 10 x 4 cells per well. After addition of complete medium, the cells were incubated in a 37℃5% CO2 incubator for 24 hours until the bottom of the well plate was completely confluent.
(2) Scratches were made with the aid of a 10 μl gun head and ruler, with the direction of the scratches perpendicular to the marking line. A platinum wire of 0.2 mm is taken as a lead, placed into a culture medium and fixed on the edge of the pore plate by using an insulating tape.
(3) The SS-L303SPD linear power supply is used for connecting two ends of the lead, and electric stimulation is performed. The cells were subjected to electrical stimulation by applying constant currents of 1.5V, 3.0V, 4.5V and 6.0V, 2 wells were placed in each group, and the cells were subjected to electrical stimulation for 15 minutes per day, while maintaining culture in DMEM medium, and observing cell migration conditions of 0 h, 24h and 48 h, respectively.
The cells treated with electrical stimulation were observed under a microscope and quantitatively analyzed. It is seen from the cell scratch experiment that the electrical stimulation has a remarkable promotion effect on the migration of C2C12 cells. According to FIG. 8, C shows that at 48h, the cell migration effect was most pronounced in group 4.5V and the scratch was substantially healed.
Example 5
The electro-stimulated cell protocol of the conductive hydrogel prepared in example 1 is shown in fig. 2, and the specific operation steps are as follows:
(1) Parallel lines were drawn on the bottom of the 12-well plate using a mark pen, ensuring that the parallel line spacing was between 0.5-1 cm. L929 cells in the logarithmic growth phase are digested with trypsin into single cell suspension, inoculated into 12-well plates, and 4 x 10≡4 cells are seeded into each well. After addition of complete medium, the cells were incubated in a 37℃5% CO2 incubator for 24 hours until the bottom of the well plate was completely confluent.
(2) Scratches were made with the aid of a 10 μl gun head and ruler, with the scratch direction perpendicular to the mark line. A platinum wire of 0.2 mm was used as a wire and placed in a culture medium and fixed to the edge of the well plate with an insulating tape.
(3) The SS-L303SPD linear power supply is used for connecting two ends of the lead, and electric stimulation is performed. The electrical stimulation was performed at constant currents of 0.3 mA, 0.5 mA, 0.8 mA, 1.0 mA, 2 replicate wells per group, 15 minutes per day. During this period, the culture was performed in DMEM medium, and cell migration was observed at positions 0h, 24, h, 48, h, 72, h, respectively.
The cells treated with electrical stimulation were observed under a microscope and quantitatively analyzed. It was found by cell scratch experiments that electrical stimulation had a significant effect on promoting migration of L929 cells. According to the D in FIG. 8, at the time of 72 h, the cell migration effect of the 0.5 mA group was most remarkable, and the scratch area was minimum.
Example 6
The scheme of electrically stimulating cells using the conductive hydrogel prepared in example 1 is shown in fig. 2, and the specific procedure is as follows:
(1) Parallel lines were drawn on the bottom of the 12-well plate using a mark pen with a parallel line pitch controlled to be 0.5-1 cm. HUVEC cells in the logarithmic growth phase were trypsinized into single cell suspensions, inoculated into 12-well plates, and 4X 10≡4 cells were seeded into each well. After addition of complete medium, the cells were incubated in a 37℃5% CO2 incubator for 24 hours until the bottom of the well plate was completely confluent.
(2) With the aid of a ruler, scratches were made with a 10 μl gun head, the direction of the scratches being perpendicular to the marking lines. A platinum wire of 0.2 mm was used as a wire and placed in a culture medium and fixed to the edge of the well plate with an insulating tape.
(3) The SS-L303SPD linear power supply is used for connecting two ends of the lead, and electric stimulation is performed. The electrical stimulation was performed at constant currents of 0.3 mA, 0.5 mA, 0.8 mA, 1.0 mA, 2 replicate wells per group, 15 minutes per day. During this period, the cells were cultured in DMEM medium, and the migration of the cells was observed at positions 0 h, 24, h, and 48, h, respectively.
The electrically stimulated cells were observed under a microscope and quantitatively analyzed. It can be seen from the cell scratch experiments that the electrical stimulation has a remarkable effect on promoting the migration of HUVEC cells. According to FIG. 8E, it is shown that at 48 h, the 0.5 mA group of cells migrated optimally and the scratch healed substantially.
Example 7
A protocol for electrically stimulating cells using the electrically conductive hydrogel prepared in example 1 is shown in fig. 2, and a specific electrical stimulation protocol includes the following steps:
(1) Parallel lines were drawn on the bottom of the 12-well plate using a mark pen, and the parallel line pitch was controlled between 0.5 and 1 cm. C2C12 cells in the logarithmic growth phase were trypsinized into single cell suspensions and inoculated into 12 well plates, each well being seeded with 4 x 10 x 4 cells. After addition of complete medium, the cells were incubated in a 37 ℃ 5% CO2 incubator for 24 hours until the bottom of the well plate was completely confluent.
(2) Scratches were made along a ruler with a10 μl gun head, the direction of the scratches being perpendicular to the marking lines. A platinum wire of 0.2mm was used as a wire and placed in a culture medium and fixed to the edge of the well plate with an insulating tape.
(3) The SS-L303SPD linear power supply is used for connecting two ends of the lead, and electric stimulation is performed. The electrical stimulation was performed with constant currents of 0.3 mA, 0.5 mA, 0.8 mA, 1.0 mA, 2 replicate wells per group, 15 minutes per group per day. During this period, the cells were cultured in DMEM medium, and the migration of the cells was observed at positions 0h, 24, h, and 48, h, respectively.
The cells electrically stimulated in this example were observed under a microscope and quantitatively analyzed. It is seen from the cell scratch experiment that the electrical stimulation has a certain effect on promoting the migration of the C2C12 cells. According to FIG. 8, F shows that at 48 h, cell migration is optimal under electrical stimulation conditions of 0.8 mA and the scratch heals substantially.
Example 8
The scheme of electrically stimulating cells using the conductive hydrogel prepared in example 1 is shown in fig. 2, and the specific procedure is as follows:
(1) Parallel lines were drawn on the bottom of the 12-well plate using a mark pen, ensuring that the distance between the parallel lines was maintained in the range of 0.5-1 cm. HUVEC cells in the logarithmic growth phase are digested by trypsin into single cell suspension, and then inoculated into 12-well plates, and 4 x 10 times 4 cells are respectively planted in each well. After addition of complete medium, the cells were incubated in a 37℃5% CO2 incubator for 24 hours until the cells completely covered the bottom of the well plate.
(2) Scratches were made with the aid of a 10 μl gun head and ruler, ensuring that the scratch direction was perpendicular to the mark line. A platinum wire of 0.2 mm was used as a wire and placed in a culture medium and fixed to the edge of the well plate with an insulating tape.
(3) The SS-L303SPD linear power supply is used for connecting two ends of the lead, and electric stimulation is performed. Both ends of the wire were electrically stimulated using the optimal constant current determined in example 3, with 2 replicate wells per group, for 15 minutes per day. During this period, the culture was performed using DMEM medium or PWTB hydrogel extract, and cell migration was observed at 0 h, 24, h, 48, h, respectively.
The cells treated with the electrical stimulation of this example were observed under a microscope and quantitatively analyzed. The analysis result of the cell scratch experiment in fig. 9 shows that the electrical stimulation combined with the hydrogel can significantly improve the cell migration effect, thereby effectively promoting wound healing.
Example 9
A protocol for applying the electrically conductive hydrogel prepared in example 1 to an electrically stimulated wound model is shown in fig. 3. The specific scheme comprises the following steps:
(1) Female BALB/C mice of 6 weeks old and average body weight of 18 g were placed in an environment at constant temperature (23±2) ° C and humidity of 50% -60%, following a circadian rhythm of 12 hours light-12 hours darkness, and were free to drink and eat.
(2) After anesthetizing the mice, a 9mm full-thickness circular incision was made on the back with a scalpel. The blank group received no treatment. For the Electrical Stimulation (ES) group, a linear power supply was placed near the wound edge, ES treatment was performed every other day, and after 15 minutes the external power supply was removed. For the hydrogel+es group, the conductive hydrogel prepared in example 1 was applied to the wound and the treatment was performed every other day, each time by applying the hydrogel according to the electrical stimulation parameters of example 5 for 15 minutes.
By observing the change of wound area of the wound in the present embodiment (as shown in fig. 10), it can be seen that the wound contraction effect of the conductive hydrogel dressing group is better than that of the control group. The hydrogel dressing in combination with ES exhibited a more pronounced wound healing effect compared to the hydrogel or ES-treated group alone. This suggests that ES therapy has a positive effect on wound healing, but there is still room for improvement. In contrast, the wound surface of the hydrogel+ES group can be completely closed, and the wound surface repair rate of the hydrogel+ES group has a remarkable difference compared with that of a conductive hydrogel group without ES, so that the synergistic effect between the conductive hydrogel and the ES is further proved to effectively accelerate the healing of wounds.
Example 10
The conductive hydrogel prepared in application example 1 is used for monitoring human body movement and preventing wound tearing, and the specific monitoring scheme comprises the following steps:
(1) The conductive hydrogel of example 1 was adhered to each joint of the human body and fixed to both ends of the hydrogel with a wire clip.
(2) The precise source measuring unit is connected with the wire clamp to form a closed loop, and the relative resistance change of the conductive hydrogel is measured.
The change in the resistance signal of this embodiment is recorded by a precision source measurement unit. Fig. 11 shows that the conductive hydrogel sensor is connected to the integrated system, and records the resistance change of the hydrogel caused by strain during the movement of the human body, so as to monitor the movement condition of the human body and avoid secondary tearing of the wound. In addition, due to its excellent adhesion property, the hydrogel sensor can be tightly adhered to the skin of a human body, and can accurately transmit an electric signal even in the case of severe exercise or repeated deformation.
Example 11
A protocol for monitoring wound healing process using the conductive hydrogel prepared in example 1 is shown in fig. 4, and the specific monitoring protocol includes the following steps:
(1) Male SD rats, 8 weeks old and 300 grams average body weight, were placed in an environment at constant temperature (23+ -2) deg.C and humidity of 50% -60%, following a 12-hour light-12-hour dark circadian rhythm, with free drinking and eating. After anesthetizing the rat, an 8 mm circular incision was made with a scalpel at the left knee joint. The blank group received no treatment.
(2) The conductive hydrogel prepared in example 1 was placed on the wound of the left knee joint and the intact skin surface of the right knee joint of the rat, and connected to form a closed loop according to the sensing test scheme of example 8. The left knee joint of the rat is assisted to move, the amplitude is constantly controlled to be 45 degrees, and the relative resistance change of the conductive hydrogel is recorded by a precise source measurement unit.
(3) After one week, when the left knee joint wound of the rat is about to heal, the test was performed again according to step (2) of example 8.
(4) After two weeks, when the left knee joint wound of the rat was completely healed, the test was performed again according to step (2) of example 8.
(5) A piece of pig skin and silica gel with the length of 50mm, the width of 15 mm and the thickness of 10mm are prepared respectively, a surgical knife is used for carrying out shallow scratch on the back of a sample, so that no penetration of materials is ensured, the front of the sample is maintained to be complete, and a deep wound is formed on the back of the sample and is used for simulating a healing bad wound with the surface of the skin closed and the deep layer not healed.
(6) The conductive hydrogel prepared in example 1 was covered on the surface of pigskin and silica gel, the pigskin and silica gel were fixed on a manipulator, and the pulse width modulation of the manipulator was set to move from 1500 to 2500 with a time interval of 1200 ms.
The resistance signal change in this embodiment was recorded by a precision source measurement unit, and the result is shown in fig. 12. Fig. 12 (B) and (C) show a range of >10% for the resistance signal of the wound, 5-10% for normal skin or healed wound represented by fig. 12 (a) and (D), and <5% for surface healing but still damaged skin inside. Therefore, the scheme can be used for monitoring the wound healing process and the healing quality, assisting doctors in diagnosing and helping patients to better know the conditions of the patients.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, and various modifications and variations may be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.